Surgical intervention is currently the most common treatment for virtually all types of solid tumors. Siegel et al. (2012) CA Cancer J. Clin. 62:220-41; DeSantis et al. (2014) CA Cancer J. Clin. 64:252-71. A successful outcome is therefore contingent upon the complete removal of all cancer cells from both the affected primary organ and from potential metastatic sites during surgery. Vahrmeijer et al. (2013) Nat. Rev. Clin. Oncol. 10:507-18. Contrast agents that target specific biomarkers in cancers can be used as intra-operative contrast agents to guide surgical resection of solid tumors in order to improve treatment outcome. Miwa et al. (2014) J. Orthop. Res. 32:1596-601; Fujita (2012) J. Am. Coll. Surg. 215:591. Among the diverse imaging modalities, optical based techniques utilizing fluorescent contrast agents have great potential. Rudin and Weissleder (2003) Nat. Rev. Drug Discov. 2:123-31; Bednar et al. (2007) Expert Opin. Drug Discov. 2:65-85. Indocyanine green (ICG), fluorescein, methylene blue, and 5-aminolevuliric acid (5-ALA) are all non-targeted contrast agents that are currently approved as injectable enhancers for the visualization of various tumors. Schaafsma et al. (2011) J. Surg. Oncol. 104:323-32; Tanaka et al. (2006) Ann. Surg. Oncol. 13:1671-81. In addition, several targeted contrast agents are in various stages of clinical development. Kovar et al. (2007) Anal. Biochem. 367: 1-12. Notably, an FITC probe that targets folate receptor-α was used in a clinical trial to demonstrate the value of intraoperative fluorescence-guided surgery (FGS) for the treatment of ovarian cancer. van Dam et al. (2011) Nat. Med. 17:1315-9. Additionally, other tumor-targeting agents, such as Chlorotoxin-Cy5.5, have been validated for optical imaging of malignant cancer cells using various mouse models of cancer. The mechanism of tumor selectivity for this agent is not, however, well understood. Veiseh et al. (2007) Cancer Res. 67:6882-8.
An alternative approach to general tumor-targeted contrast agents is the use of so called “smart probes” that only produce or accumulate a signal in tumor tissues when acted upon by an enzyme activity that is associated with the tumor or surrounding margins. One useful strategy in smart probe design is to make probes that produce signal when cleaved by a protease. Because proteases play significant roles in tumor growth and metastasis as well as in diverse pathologies such as fibrosis, inflammation, osteoporoses, and arthritis, contrast agents that are activated by proteases could prove valuable for the detection and treatment of many diseases. Turk (2006) Nat. Rev. Drug Discov. 5:785-99; Drag and Salvesen (2010) Nat. Rev. Drug Discov. 9:690-701.
A number of probes for tumor imaging applications have targeted the matrix metallo proteases (MMPs) due to their reported roles in angiogenesis and tumor growth. This includes both small molecule and large polymer-based probes that produce a signal upon cleavage as well as masked cell-penetrating peptides that accumulate inside cells when cleaved by an MMP. As an alternative to the MMPs, the cysteine cathepsins are important regulators of various aspects of tumorigenesis. Shree et al. (2011) Genes Dev. 25:2465-79. These proteases are also highly expressed and activated in many cells that regulate the intrinsic inflammatory response. Mohamed and Sloane (2006) Nat. Rev. Cancer 6:764-75. In general, cysteine cathepsin activities are elevated in virtually all solid tumors due to increased infiltration of immune cells. Mitchem et al. (2013) Cancer Res. 73:1128-41; McIntyre and Matrisian (2003) J. Cell. Biochem. 90:1087-97; Fonovic and Bogyo (2007) Curr. Pharm. Des. 13:253-61; Gocheva et al. (2010) Genes Dev. 24:241-55. The cysteine cathepsins have therefore been targeted in the design of tumor-specific contrast imaging agents. Such agents include fluorescent activity-based probes that covalently modify the cathepsins during turnover (Verdoes et al. (2013) J. Am. Chem. Soc. 135:14726-30; Lee and Bogyo (2010) ACS Chem. Biol. 5:233-43; Blum et al. (2005) Nat. Chem. Biol. 1:203-9; Blum et al. (2007) Nat. Chem. Bio. 3:668-677; Verdoes et al. (2012) Chem. Biol. 19:619-28), a range of high and low molecular weight quenched substrate probes (Watzke et al. (2008) Angew. Chem. Int. Ed. Engl. 47:406-9; Hu et al. (2014) Angew. Chem. Int. Ed. Engl. 53:7669-73), and fluorogenic turn-on substrate probes (Kisin-Finfer et al. (2014) Bioorg. Med. Chem. Lett. 24:2453-8; Chowdhury et al. (2014) J. Med. Chem. 57:6092-104; Fujii et al. (2014) Bioconjug. Chem. 25:1838-46). Although all of the reported protease-triggered smart probes have proven useful for imaging of tumor margins in mouse models of cancer (Verdoes et al. (2013) J. Am. Chem. Soc. 135:14726-30; Hu et al. (2014) Angew. Chem. Int. Ed. Engl. 53:7669-73; Mito et al. (2012) Cancer 118:5320-30), all have limitations in terms of tumor contrast and none have been used with clinically-approved imaging instrumentation. Furthermore, most have only been validated using simple graft models of cancer in which large tumors are imaged at or near the skin surface. Therefore, the optimization of a targeted contrast agent with enhanced contrast for multiple tumor types and that could be used with existing clinical instrumentation within the confines of existing surgical workflows would be transformative to many surgical procedures.
Methods and materials for the imaging of cells containing active proteases such as cathepsins are disclosed in U.S. Patent Application Publication No. 2007/0036725. Radiolabeled activity-based probes useful in the radiolabeling of target enzymes, including cathepsins, in vivo are disclosed in U.S. Patent Application Publication No. 2009/0252677. In each of cases, the probes employ an ester-linked acyloxymethyl ketone (AOMK) reactive group to modify the protease active site covalently. Non-peptidic activity-based fluorescent probes are disclosed in PCT International Publication No. WO 2012/118715.
PCT International Publication No. WO 2014/145257 discloses quenched ABPs comprising an ether-linked, 2,3,5,6-tetrafluoro-substituted phenoxymethyl ketone (PMK) leaving element. The detectable component of the disclosed ABPs remain covalently attached to their target protease after enzymatic turnover.
U.S. Patent Application Publication No. 2014/0301950 discloses imaging agents comprising a dark quencher, an amino acid backbone, a fluorophore, 6-aminohexanoic acid, aminoethoxyethoxyacetyl spacers, and a methoxypolyethylene glycol (mPEG) chain. The agents are purportedly cleaved by cathepsins to generate a fluorescent signal and thus to image diseased cells. The technology is directed at identifying diseased cells at or near the surface of a tissue.
Despite these disclosures, there remains a need in the field for novel activity-based contrast agents that have high cellular uptake, that target a broad spectrum of animal proteases, and that offer increased sensitivity of detection at a variety of wavelengths, particularly at wavelengths capable of penetrating diseased tissue.